[Show abstract][Hide abstract] ABSTRACT: Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30 min of ischemia followed by 2 h of reperfusion (IR) or IP prior to 30 min ischemia and 2 h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63 mmHg), dp/dt (max) (893 versus 1,027 mmHg/s), and aortic flow (0.9 versus 1.8 ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120 min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as β-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin 1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild-type (W) and Trx1 transgenic (Trx1(Tg/+)) mice were randomized into W sham (WS), Trx1(Tg/+) sham (TS), WMI, and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS, p-GSK-3β, HIF-1α, β-catenin, VEGF, Bcl-2, and survivin expression in TMI compared to WMI. Echocardiography performed 30days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12, and subsequent activation of Akt/GSK-3β/β-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.
Journal of Molecular and Cellular Cardiology 11/2010; 50(1):239-47. DOI:10.1016/j.yjmcc.2010.11.002 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study evaluated the reversal of diabetes-mediated impairment of angiogenesis in a myocardial infarction model of type 1 diabetic rats by intramyocardial administration of an adenoviral vector encoding thioredoxin-1 (Ad.Trx1). Various studies have linked diabetes-mediated impairment of angiogenesis to dysfunctional antioxidant systems in which thioredoxin-1 plays a central role.
Ad.Trx1 was administered intramyocardially in nondiabetic and diabetic rats immediately after myocardial infarction. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. Myocardial function was measured by echocardiography 30 days after the intervention. The Ad.Trx1-administered group exhibited reduced fibrosis, oxidative stress, and cardiomyocyte and endothelial cell apoptosis compared with the diabetic myocardial infarction group, along with increased capillary and arteriolar density. Western blot and immunohistochemical analysis demonstrated myocardial overexpression of thioredoxin-1, heme oxygenase-1, vascular endothelial growth factor, and p38 mitogen-activated protein kinase-beta, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-alpha, in the Ad.Trx1-treated diabetic group. Conversely, we observed a significant reduction in the expression of vascular endothelial growth factor in nondiabetic and diabetic animals treated with tin protoporphyrin (SnPP, a heme oxygenase-1 enzyme inhibitor), even after Ad.Trx1 therapy. Echocardiographic analysis after 4 weeks of myocardial infarction revealed significant improvement in myocardial functional parameters such as ejection fraction, fractional shortening, and E/A ratio in the Ad.Trx1-administered group compared with the diabetic myocardial infarction group.
This study demonstrates for the first time that impairment of angiogenesis and myocardial dysfunction can be regulated by Ad.Trx1 gene therapy in streptozotocin-induced diabetic rats subjected to infarction.
[Show abstract][Hide abstract] ABSTRACT: Hyperglycemia impairs angiogenesis in response to ischemia, leading to ventricular remodeling. Although the effects of overexpressing angiogenic growth factors have been studied in inducing angiogenesis, the formation of functional vessels remains a challenge. The present study evaluates the reversal of diabetes-mediated impairment of angiogenesis in the infarcted diabetic rat myocardium by proangiogenic gene therapy.
Ad*VEGF and Ad*Ang1 were intramyocardially administered in combination immediately after myocardial infarction to nondiabetic and diabetic rats. Ad*LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. The myocardial function was measured by echocardiography 30 days after the intervention.
We observed reduced fibrosis and increased capillary/arteriolar density along with reduced ventricular remodeling, as assessed by echocardiography in the treated diabetic animals compared with the nontreated diabetic controls. We also observed increased phosphorylated mitogen-activated protein kinase-activated protein kinase-2, 2 days after the treatment and increased expression of vascular endothelial growth factor (VEGF), Flk-1, angiopoietin-1 (Ang-1), Tie-2, and survivin, 4 days after treatment in the diabetic animals. Gel shift analysis revealed that the combination gene therapy stimulated the DNA binding activity of nuclear factor-kappaB in the diabetic animals.
Our preclinical data demonstrate the efficacy of coadministration of adenoviral VEGF and Ang-1 in increasing angiogenesis and reducing ventricular remodeling in the infarcted diabetic myocardium. These unique results call for the initiation of a clinical trial to assess the efficacy of this therapeutic strategy in the treatment of diabetes-related human heart failure.
[Show abstract][Hide abstract] ABSTRACT: Myocardial ischemia and cardiac dysfunction have been known to follow ischemic heart diseases (IHDs). Despite a plethora of conventional treatment options, their efficacies are associated with skepticism. Cell therapies harbor a promising potential for vascular and cardiac repair, which is corroborated by adequate preclinical evidence. The underlying objectives behind cardiac regenerative therapies subsume enhancing angiomyogenesis in the ischemic myocardium, ameliorating cellular apoptosis, regenerating the damaged myocardium, repopulating the lost resident myocardial cells (smooth muscle, cardiomyocyte, and endothelial cells), and finally, decreasing fibrosis with a consequent reduction in ventricular remodeling. Although-cell based cardiomyoplasty approaches have an immense potential, their clinical utilization is limited owing to the increased need for better candidates for cellular cardiomyoplasty, better routes of delivery, appropriate dose for efficient engraftment, and better preconditioning or genetic-modification strategies for the progenitor and stem cells. Mesenchymal stem cells (MSCs) have emerged as powerful candidates in mediating myocardial repair owing to their unique properties of multipotency, transdifferentiation, intercellular connection with the resident cardiomyocytes via connexin 43 (Cx43)-positive gap junctions in the myocardium, and most important, immunomodulation. In this review, we present an in-depth discussion on the complexities associated with stem and progenitor cell therapies, the potential of preclinical approaches involving MSCs for myocardial repair, and an account of the past milestones and ongoing MSC-based trials in humans.
[Show abstract][Hide abstract] ABSTRACT: The last few decades have seen significant advancement in the therapy of Ischemic Heart Diseases (IHD). This is a direct outcome of the increasing knowledge of the molecular mechanisms involved during an ischemic insult of the myocardium. Even then there is still a major unmet need for better strategies or drug therapies to reduce ventricular remodeling and improve post-ischemic myocardial function. The ex-vivo isolated working heart model and the in vivo myocardial infarction model are the best known techniques to elucidate the contribution of a drug therapy to confer cardioprotection in the event of an ischemic insult/reperfusion. Our review aims to provide an insight into the state of the art techniques that lay the foundations for cardiovascular drug discovery and present the prospects for further development from a preclinical perspective. The first section of the review provides an overview of the rat/mouse ex-vivo and in vivo models of myocardial ischemia. The following section will then present various applications of these clinically relevant models in characterizing cardiac functions, screening for drugs and identifying the drug induced changes in cardiac functions. Finally the role of these models in drug development is discussed with respect to functional relevance of drug treatment on heart rate, aortic flow, coronary flow, infarct size and the mechanisms by which these drugs promote myocardial protection. This review may serve as a basic knowledge for researchers who intend to study the efficacy of a drug in the treatment of ischemic heart diseases.
Current Drug Discovery Technologies 01/2009; 5(4):269-78. DOI:10.2174/157016308786733555
[Show abstract][Hide abstract] ABSTRACT: Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr(3+)) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia+NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia+NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia+NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia+NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.
[Show abstract][Hide abstract] ABSTRACT: Moderate consumption of wine has been associated with decreased risk of cardiovascular events. Recently we have shown that white wine is equally as cardioprotective as red wine. However, unlike resveratrol (polyphenol in red wine), the white wine component, n-tyrosol [2-(4-hydroxyphenyl)ethanol] has not been explored for its cardioprotective effect and mechanism of action. Therefore, the present study was designed to evaluate the effect of tyrosol treatment (5 mg/kg/day for 30 days) on myocardial ischemic stress in a rat in vivo model of Myocardial Infarction (MI) and to identify key molecular targets involved in this mechanism. MI was induced by Left Anterior Descending (LAD) coronary artery ligation. Reduced infarct size (32.42 vs 48.03%) and cardiomyocyte apoptosis (171 vs 256 counts/100 HPF) were observed along with improvement in the myocardial functional parameters such as LVIDs (5.89 vs 6.58 mm), ejection fraction (51.91 vs 45.09%), and fractional shortening (28.46 vs 23.52%) as assessed by echocardiography in the tyrosol-treated animals when compared to the nontreated controls. We have also observed significant increase in the phosphorylation of Akt (1.4-fold), eNOS (3-fold) and FOXO3a (2.6-fold). In addition, tyrosol induced the expression of longevity protein SIRT1 (3.2-fold) in the MI group as compared to the non-treated MI control. Therefore tyrosol's SIRT1, Akt and eNOS activating power adds another dimension to the white wine research, because it adds a great link to the French paradox. In conclusion these findings suggest that tyrosol induces myocardial protection against ischemia related stress by inducing survival and longevity proteins that may be considered as anti-aging therapy for the heart. However, human intervention studies would be necessary before establishing any recommendations about dietary habits for tyrosol intake or administration of dietary supplements containing tyrosol.
Journal of Agricultural and Food Chemistry 11/2008; 56(20):9692-8. DOI:10.1021/jf802050h · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies on the protection afforded by moderate wine consumption against cardiovascular diseases have focused mainly on the activity of red wine in view of its high content of antioxidants, especially polyphenols. White wine lacks polyphenols, but it contains other compounds such as hydroxycinnamic acids (caffeic acid) and monophenols (tyrosol), which are known to have antioxidant properties. Therefore, this study was designed to examine the effect of white wine in myocardial ischemic-reperfusion injury. The experimental rats were gavaged with white wine (Soave Suavia "Le Rive" 2004) at a dosage of 6.5 mL/(kg.rat.day) for 30 days. Rats were divided into four groups: control sham (CS), wine-treated sham (WS), control ischemia (I)/reperfusion (R) (CIR), and wine + IR (WIR). All the rats in both IR groups underwent 30 min occlusion of the left anterior descending coronary artery followed by 8, 24 h, and 30 days of reperfusion (R). Significant reduction in infarct size (21 vs 39%, n = 6), cardiomyocyte (274 vs 384 counts/100 HPF, n = 6), and endothelial cell apoptosis (387 vs 587 counts/100 HPF) was observed in WIR as compared with CIR after 24 h of reperfusion. Echocardiography demonstrated significant increased fractional shortening (32 vs 22%) and ejection fraction (60 vs 44%) following 30 days of reperfusion in WIR rats compared to CIR ( n = 6). In addition, increased phosphorylation of AKT, Foxo3a, and eNOS were found in WS and WIR, as compared to their respective controls. The gel-shift analysis demonstrated significant upregulation of DNA binding activity of NF-kappaB in the white wine-treated groups. This report demonstrated for the first time that the white wine mediated cardioprotection in ischemic reperfused myocardium is through the PI-3kinase/Akt/FOXO3a/e-NOS/NF-kappaB survival pathway.
Journal of Agricultural and Food Chemistry 08/2008; 56(15):6733-9. DOI:10.1021/jf801473v · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial dysfunction and impaired angiogenesis constitute a hallmark of hypercholesterolemia. This study was designed to examine the effects of resveratrol, an antioxidant with lipid-lowering properties similar to those of statins, on neovascularization along with caveolar interaction with proangiogenic molecules in hypercholesterolemic rats. Animals were divided into: rats maintained on a normal diet (control group); rats maintained on a 5% high-cholesterol diet for 8 weeks (HC group); and rats maintained on a 5% high-cholesterol diet for 8 weeks and administered resveratrol (20 mg/kg) orally for 2 weeks (HCR group). Myocardial infarction was induced by ligating the left anterior descending artery. Herein we examined a novel method for stimulating myocardial angiogenesis by pharmacological preconditioning with resveratrol at both the capillary and arteriolar levels and the potential role of hemeoxygenase-1, endothelial nitric oxide synthase and caveolin-1 in mediating such a response. We also investigated the functional relevance of such treatment by assessing whether the induced neovascularization can help preserve left ventricle-contractile functional reserve in the setting of a chronic hypercholesterolemic condition. Four weeks after sham surgery and left anterior descending artery occlusion, rats underwent echocardiographic evaluation, which revealed improvement in ejection fraction and fractional shortening in the HCR group compared with the HC group. Left ventricular tissue sections displayed increased capillary and arteriolar density in the HCR group compared with the HC group. Western blot analysis revealed downregulation of vascular endothelial growth factor and hemeoxygenase-1 and increased association of caveolin-1 eNOS in the HC group, decreasing the availability of eNOS to the system; which was reversed with resveratrol treatment in the HCR group. This study was further validated in cardiac-specific hemeoxygenase-1-overexpressed mice assuming molecular cross-talk between the targets. Hence, our data identified potential regulators that primarily attenuate endothelial dysfunction by resveratrol therapy in hypercholesterolemic myocardium.
Free Radical Biology and Medicine 08/2008; 45(7):1027-34. DOI:10.1016/j.freeradbiomed.2008.07.012 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examined the hypothesis that the ubiquitin proteasome system (UPS) degrades proteins damaged by exposure to hyperglycemia. Experimental hyperglycemia was induced in male rats by treatment with streptozotocin. After 30 days, echocardiography confirmed the presence of cardiomyopathy as ejection fraction, fractional shortening, and diastolic function (E/A ratio) were decreased, and chamber diameter was increased in hyperglycemic animals. Proteasome non-ATP-dependent chymotryptic activity was increased over 2-fold in hyperglycemic hearts, but the ATP-dependent activity was decreased and levels of ubiquitinated proteins were increased. Protein levels of the PA28alpha of the 11S-activator ring were increased by 128% and the PA28beta subunit increased by 58% in the hyperglycemic hearts. The alpha3 subunit of the 20S-proteasome was increased by 82% while the catalytic beta5 subunit was increased by 68% in hyperglycemic hearts. Protein oxidation as indicated by protein carbonyls was significantly higher in hyperglycemic hearts. These studies support the conclusion that the UPS becomes dysfunctional during long term hyperglycemia. However, 11S-activated proteasome was increased suggesting a response to oxidative protein damage and a potential role for this form of the proteasome in a cardiac pathophysiology.
Journal of Molecular and Cellular Cardiology 04/2008; 44(3):618-21. DOI:10.1016/j.yjmcc.2007.12.009 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-L-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.